Project Summary (Bourne Project) Toxin-Antitoxin (TA) modules are conserved interacting proteins that affect the survival of bacterial cells either by promoting the type of growth found in chronic infections, or by inducing bacterial cell death. Difficult to treat and chronic infections are increasingly prevalent, and the Centers for Disease Control estimate at least 2 million antibiotic-resistant infections occur in the United States each year at a cost of up to $20-billion in added healthcare expenses. TA modules enhance E. coli survival during antibiotic treatments, called antibiotic tolerance, which could reduce the efficacy of antimicrobial treatments. The current proposal focuses on TA modules in Pseudomonas aeruginosa, an opportunistic human pathogen of serious concern due to the prevalence of chronic and antibiotic resistant infections. Despite high variability between clinical isolates, this bacteria contains two highly conserved TA modules, 1) a RelBE/ParDE gyrase-inhibiting family module, and 2) a HigBA ribosome-inhibiting module recently shown to affect virulence production in this organism. One-third of clinical P. aeruginosa isolates contain an additional conserved ParDE system. TA modules are typically expressed as an interacting pair, thus rendering them inactive. Extracellular signals can trigger degradation of the antitoxin, releasing the toxin within the cytosol to interact with cell machinery and leading to alteration of the rate of cell replication or translation. Currently, there are fundamental gaps in our knowledge of TA module functions that prevent translation into clinical settings, including the regulation of their activation and the effect of sustained interaction of toxins with their targets. The current proposal will make significant vertical advances in the field of bacterial TA biology by directly addressing these gaps. The overall objective of the current proposal is to determine the interaction of toxins found in P. aeruginosa with their cellular targets, particularly those inhibiting DNA gyrase, and to determine the molecular interactions of the antitoxins that result in activation of these type II TA modules. The central hypothesis is that the same toxin molecule can instigate both cellular dormancy and cell death as depending on its individual potency and the length of interaction with the cellular target, and that degradation of the antitoxin is mediated by different cellular proteases depending on the specific environmental trigger. Aim 1 will pursue the structure of the toxins interacting with DNA gyrase and will measure their potency using in vitro assays. Aim 2 will determine the cellular proteins that interact with the antitoxins to mediate its degradation using a genetic interaction screening platform. Aim 3 will establish the mRNA and protein levels of the three TA modules under different environmental conditions. Successful completion of these aims will determine critical steps in TA biology. In addition, mapping the cellular proteases that control the specific responses will be key in understanding the role of TA modules in bacterial physiology. These studies will directly impact the field by defining fundamental regulatory mechanisms in P. aeruginosa that contribute to antibiotic tolerance and chronic infections, thereby revealing new or synergistic antibacterial targets.